The arthropod-borne viruses within the genus Flavivirus of the Flaviviridae family are important human pathogens, causing outbreaks of meningoencephalitis in humans in many regions of the world with fatality rates between 10 and 50%. Flaviviruses are transmitted in nature to various mammals and birds through the bite of an infected mosquito or tick and include mosquito-borne Dengue, WN, Japanese encephalitis, SLE, and tick-borne encephalitis viruses. WN and SLE are endemic in North America, and WN is the major cause of viral encephalitis in the USA. During the 1999-2011 outbreaks, there were at least 31,414 reported human cases of WN illness that resulted in 1263 deaths. Since 2004, there was a steady decrease in incidence of WN disease; then in 2012, WN virus resurgence produced 5,674 human infections including 2,873 cases of neuroinvasive disease and 286 deaths. The upsurge of human WN infections suggests that the US can expect periodic WN outbreaks in the coming years. No established specific antiviral therapy or a licensed human vaccine is available to date to treat and prevent SLE and WN diseases. Tick-borne encephalitis is a severe neurologic disease affecting thousands of people throughout Eurasia. Despite the use of formalin-inactivated vaccines in endemic areas, an increasing incidence of TBEV during the past 2 decades emphasizes the need for an alternative vaccine that will induce a more durable immunity and protection against TBEV. In an effort to develop the efficacious live attenuated vaccines against neurotropic flaviviruses, we explored several strategies, and progress toward this goal in the past year is reviewed below. The live attenuated TBEV or WN vaccine candidates are being developed in the Neurotropic Flaviviruses Section of the LID using a strategy based on the chimerization of a neurovirulent TBEV or WN with a non-neuroinvasive dengue-4 flavivirus (DEN4) and the introduction of attenuating mutations or targets for cellular microRNAs (miRNAs). The introduction of a 30 nucleotide deletion in the 3non-coding region (3NCR) of chimeric TBEV/DEN4 or WN/DEN4 genome that contains the structural protein genes of a highly virulent TBEV or WN has a significant attenuating effect on neurovirulence, neuroinvasiveness, and pathogenesis in adult mice. However, in a more susceptible animal model such as newborn or immunodeficient mice or monkeys, TBEV/DEN4d30 exhibited a high level of neurovirulence in the CNS compared to that observed for widely used yellow fever 17D vaccine virus. Further attenuation of TBEV/DEN4 was required since a live vaccine against neurotropic virus should be fully restricted for replication in the CNS. The discovery of miRNAs, small noncoding RNAs that regulate the expression of cellular genes, has enabled us to use a novel strategy to modulate virus tropism and selectively attenuate its pathogenesis. We explored the ability of the CNS-expressed microRNAs to modulate the neurotropism and pathogenesis of flaviviruses. The inclusion of a single target for the brain tissue-expressed miRNAs into the TBEV/DEN4 genome was sufficient to prevent the development of otherwise lethal encephalitis in adult mice. However, we found that the efficacy of miRNA-mediated inhibition of virus replication in the immature CNS of suckling mice depended on the genetic stability of the miRNA-targeted virus that can revert to a virulent phenotype. In FY2012, we demonstrated that multiple site miRNA-targeting of flavivirus genome in the 3NCR strongly attenuated the virus neurovirulence and pathogenesis in the developing mouse CNS. Importantly, virus escape from miRNA-mediated suppression occurs exclusively through the deletion of inserted miRNA-targets and results in the loss of the viral genome sequence located between the two most distant miRNA targets. These findings offer a general strategy to control the virus escape and reversion to a virulent phenotype: simultaneous miRNA-targeting of the viral genome at many different functionally important regions (5NCR, ORF, and 3NCR) could prevent virus escape from miRNA-based attenuation. In FY2013, two strategies have been tested to explore whether miRNA targeting of viral genome at the ORF could attenuate virus neurovirulence: (1) the replacement of viral genome sequence with mir-124 sequence in the regions of their homology identified in E, NS3, or NS5 gene and (2) the insertion of miRNA-targets between the viral genes. With the first strategy, genome sequence replacements always led to amino acid changes in the proteins making virus recovery unpredictable or resulting in inefficient virus replication or insert instability. In the second approach, a set of miRNA-targets was introduced between the TBEV E and DEN4 NS1 genes of the TBEV/DEN4 genome. Insertion of three target copies for mir-124 miRNA or three target copies for mir-9 and mir-124 miRNAs between these genes resulted in the attenuation of virus neurovirulence for newborn mice. However, similar to the viruses carrying miRNA-targets in the 3NCR, these viruses escaped from the miRNA-mediated inhibition through the deletion of miRNA targets. To further increase the level of miRNA-controlled suppression, we explored the effect of the simultaneous miRNA-targeting of viral genome in the 3NCR and sites between the E and NS1 genes. A set of four viruses that contained three targets for mir-124 miRNA or mir-9 and mir-124 miRNAs between the E and NS1 genes and three targets for mir-124 miRNA in the 3NCR have been generated. Combining the miRNA-targeting in the ORF and 3NCR had an additive effect on reducing neurovirulence of the TBEV/DEN4 virus since no death or neurological signs were observed in newborn mice inoculated in the brain with 100 or 1000 LD50. Thus, a possible solution to eliminate the flavivirus replication in the CNS and completely prevent the emergence of escape mutants is to target the functionally essential regions of the viral genome; a deletion of the target genomic sequences located between the inserted miRNA binding sites would be lethal for the virus. In FY2013, we developed new alternative TBEV vaccine candidates using an infectious cDNA clone of a naturally attenuated, tick-borne Langat virus (LGT). This project was initiated to generate LGT-based TBEV vaccines that might provide greater humoral and cellular immunity against TBEV than our lead candidate based on the DEN4 genetic backbone. Chimeric TBEV/LGT virus bearing the structural protein genes of TBEV retained the level of peripheral virulence observed for its attenuated LGT parent in mice. Using multiple site miRNA-targeting, we abolished or restricted the TBEV/LGT replication in the brain. In FY2014, we plan to exploit hematopoietic system-specific miRNAs and limit virus dissemination in the periphery by inserting targets into the viral genome for miRNAs expressed in the cells of its primary replication: e.g., dendritic cells, monocytes, and macrophages. A live attenuated WN vaccine (WN/DEN4d30 virus) developed in the NFS was found to be well-tolerated, safe, and induced a potent and durable WN antibody response in healthy adult volunteers in Phase I clinical trials at the Johns Hopkins School of Hygiene and Public Health. The clinical, virology, and histopathology data for neurovirulence and neuropathogenesis in rhesus monkeys obtained in FY2013 suggest that the WN vaccine virus is sufficiently attenuated for the CNS as compared to YF 17D vaccine and support the further large-scale clinical trials in humans, including the at-risk group of elderly volunteers. We will continue the basic research directed toward the understanding of viral neuropathogenesis and cellular biomarkers and pathways in the antiviral response associated with infection by neurovirulent (WN) or attenuated (YF 17D and WN vaccines) flaviviruses in the nonhuman.